Objectives: Polymorphisms in α-actinin-3 (ACTN3) gene are considered to be important in the genetic predisposition to human athletic performance. Most of the activities in soccer such as jumping, striking the ball, and running are considered plyometric and explosive. Performance power during such activities depends on the strength of the muscles involved. Therefore, high muscle strength is considered crucial for soccer players. The primary objective of this study was to ascertain the association between ACTN3 R577X (rs1815739) polymorphism and sports athletism in Iranian elite male soccer players.Methods: In total, 90 top-level professional Iranian male soccer players and 200 nonathletic Iranian men from the general population participated in this case–control study. Exon 16 of ACTN3 was genotyped throughout polymerase chain reaction (PCR) followed by Restriction Fragment Length Polymorphism (RFLP) using the restriction enzyme DdeI and direct sequencing. The genotypic and allelic frequencies of R577X polymorphism in athletes were compared to the frequencies in the general population (nonathletes).Results: According to the results, the percentage distributions of 577RR and 577RX genotypes (41% and 37%, respectively) were significantly higher and lower, respectively, than that of controls (25% and 57%, respectively) (P<0.001). However, no statistically significant difference was found between allelic frequencies (P=0.20).Discussion: Our findings showed a significant association between ACTN3 genotypes and elite sport performance among Iranian male soccer players, which agrees with several previous studies.